CN108450482B - Plant growth regulator for improving stress resistance and application thereof - Google Patents
Plant growth regulator for improving stress resistance and application thereof Download PDFInfo
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- CN108450482B CN108450482B CN201810252850.9A CN201810252850A CN108450482B CN 108450482 B CN108450482 B CN 108450482B CN 201810252850 A CN201810252850 A CN 201810252850A CN 108450482 B CN108450482 B CN 108450482B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
Abstract
The invention provides a plant growth regulator for improving stress resistance and application thereof. The plant growth regulator consists of lactobacillus fermentation culture solution, has excellent thermal stability, safety and no side effect, can obviously improve the capability of resisting biological stress and abiotic stress of plants, and is further used for the plant growth regulator or the application of preparing the composition for improving the capability of resisting the biological stress of the plants.
Description
[ technical field ] A method for producing a semiconductor device
The present invention relates to a plant growth regulator and its application, in particular, it relates to a natural plant growth regulator for raising stress resistance and its application.
[ background of the invention ]
During the growth of plants, the plants are often affected by biological stress and non-biological stress, and the severe plants cause the reduction of crop yield and cause agricultural economic loss. In general, the biotic stress is caused by pathogenic infection, and infected plants gradually yellow, rot, wither and even die. Abiotic stress conditions such as high temperature, cold damage, drought, high salinity and radiation exposure can alter the growth process of plants, and excessive Reactive Oxygen Species (ROS) can be generated to cause oxidative stress, severe cases and even death.
Most currently chemical pesticides or fertilizers are applied to help plants overcome the above mentioned stresses. However, the residue of chemical pesticides or fertilizers may cause environmental pollution, and thus the development of non-chemical pesticides or fertilizers to help crops to resist adverse circumstances is an important issue.
The probiotics have long been developed, are safe and have no side effect, and the previous researches indicate that the lactic acid bacteria of specific strains have the effect of inhibiting pathogenic bacteria. For example, Lactobacillus plantarum (Lactobacillus plantarum) can inhibit growth of plant pathogenic bacteria such as Fusarium oxysporum (Xanthomonas campestris) which is a black rot disease, Fusarium oxysporum (Fusarium oxysporum) which is a wither disease, and Phytophthora delbrueckii (Phytophthora drechsleri Tucker) which is an epidemic disease; fermentation broth of Lactobacillus casei (Lactobacillus casei), Lactobacillus rhamnosus (L.rhamnosus), Lactobacillus fermentum (L.fermentum), Lactobacillus reuteri (L.reuteri) and Lactobacillus plantarum inhibit Colletotrichum (Colletotrichum gloeosporioides) causing anthracnose and Botrytis cinerea (Botrytis cinerea) causing gray mold. However, without empirical studies, it is not expected that different plants will have the ability to increase resistance to biotic and abiotic stress, and there is still a need to find better plant growth regulators.
In view of the above, there is a need for a plant growth regulator, which is beneficial to improve the plant anti-stress ability in the future, and further helps the plant resist the stress and even improve the ability of the plant to adapt to climate change.
[ summary of the invention ]
Therefore, it is an object of the present invention to provide a plant growth regulator with improved stress tolerance, which consists of a lactobacillus fermentation broth.
The invention also aims to provide the application of the lactobacillus fermentation culture solution in preparing the plant growth regulating composition for improving the stress resistance, which is used for improving the capability of resisting biological stress or abiotic stress of plants by applying the lactobacillus fermentation culture solution to the whole plants, plant parts and/or culture media of the plants.
According to the above purpose of the invention, the plant growth regulator for improving stress resistance is provided, which consists of lactobacillus fermentation culture solution.
In the above examples, the fermentation broth of Lactobacillus was derived from Lactobacillus paracasei (Lactobacillus paracasei) GMNL-32 (also known as GM-080) and was collected in the preservation center for type culture Collection of Wuhan university at Lophania marchanensis, Wuchang, Hubei, China, with the preservation number of CCTCC M204012.
In one embodiment of the present invention, the lactobacillus fermentation broth may be, for example, live or dead bacteria containing lactobacillus paracasei GMNL-32.
According to another object of the invention, the use of a lactobacillus fermentation broth for preparing a plant growth regulating composition for improving stress tolerance is provided, which comprises administering the lactobacillus fermentation broth to the whole plant, plant part and/or cultivation medium of a plant to improve the plant's ability to resist biotic or abiotic stress.
In an embodiment of the present invention, the plant may be a dicot or a monocot, for example. In one example, the dicotyledonous plant is a plant of the family Solanaceae or a plant of the family Cucumis.
In an embodiment of the present invention, the biological stress may include infection by pathogenic bacteria. In one example, the pathogenic bacteria include anthrax and pseudomonas.
In one embodiment of the present invention, the abiotic stress may be, for example, high temperature of at least 45 ℃, ultraviolet light, and drought for at least 14 days.
The plant growth regulator for improving the stress resistance consists of a fermentation culture solution of lactobacillus paracasei, has excellent thermal stability, is safe, does not have side effects, can obviously improve the capability of resisting biological stress and abiotic stress of plants, and is further used as a plant growth regulator or a composition for preparing the composition for improving the stress resistance of plants.
[ description of the drawings ]
The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings in which:
FIGS. 1A and 1B show the appearance of leaves inoculated with colletotrichum gloeosporioides (FIG. 1A) and the number of lesions per leaf histogram (FIG. 1B) after a tomato plant according to an embodiment of the present invention is administered with a plant growth regulator;
FIGS. 2A to 2D show the appearance of leaves (FIGS. 2A and 2C) and the number of lesions per leaf histogram (FIGS. 2B and 2D) of a tomato plant inoculated with Pseudomonas syringae after administration of a plant growth regulator according to an embodiment of the present invention;
FIGS. 3A and 3B show the appearance of plants heat-treated with a plant growth regulator (FIG. 3A) and a histogram of heat damage (FIG. 3B) of tomato plants according to an embodiment of the present invention;
FIGS. 4A and 4B show the appearance of plants irradiated with ultraviolet rays after administration of a plant growth regulator to tomato plants according to an embodiment of the present invention (FIG. 4A) and a histogram of thermal damage (FIG. 4B);
FIGS. 5A and 5B show the appearance of drought-treated tomato plants (FIG. 5A) and a histogram of drought damage (FIG. 5B) after administration of a plant growth regulator;
FIGS. 6A and 6B show histograms of the number of root bacteria to which a plant growth regulator is administered after a tomato plant according to an embodiment of the present invention is inoculated with a bacterial solution of Bacillus thuringiensis (FIG. 6A) or Bacillus amyloliquefaciens (FIG. 6B);
FIG. 7 shows a histogram of the hydrogen peroxide content in leaves of tomato plants according to an embodiment of the present invention given to a plant growth regulator;
FIG. 8 shows a histogram of the leaf catalase activity content of tomato plants according to an embodiment of the present invention given to a plant growth regulator;
(FIG. 9A) to (FIG. 9F) show the relative mRNA content histograms of the genes LeOPR3, LeCOI1 and LeJAZ1 after the administration of plant growth regulators to tomato plants according to an embodiment of the present invention;
[ FIG. 10A ] and [ FIG. 10B ] show the relative mRNA content histograms of the gene LePR1 after the administration of plant growth regulator to tomato plants according to one embodiment of the present invention.
[ detailed description ] embodiments
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Numerical ranges (e.g., 10% to 11% A) include upper and lower limits (i.e., 10% to 11%) unless otherwise specified; numerical ranges without lower limits (e.g., less than 0.2% B, or less than 0.2% B) are all meant to indicate that the lower limit may be 0 (i.e., 0% to 0.2%). The words used above are words of description and understanding, rather than words of limitation.
The invention provides a plant growth regulator for improving stress resistance and application thereof, wherein the plant growth regulator is composed of a lactobacillus fermentation culture solution and can obviously improve the biological stress resistance and abiotic stress resistance of plants.
In one embodiment, the Lactobacillus fermentation broth is derived from Lactobacillus paracasei (Lactobacillus paracasei) GMNL-32 (also known as GM-080). Specifically, the Lactobacillus paracasei GMNL-32 refers to a strain which is preserved in the Wuchang Lojia mountain Wuhan university culture Collection (CCTCC) in Wuhan city, Wuhan, Hubei, China at 2.19.2004 and has the preservation number of CCTCC M204012.
In the above-mentioned examples, the fermentation culture of Lactobacillus may contain, for example, viable or dead Lactobacillus paracasei GMNL-32.
When in application, the lactobacillus fermentation culture solution can be used for preparing the plant growth regulating composition for improving the stress resistance. In an embodiment of the present invention, the lactobacillus fermentation broth may be administered to the whole plant, plant part and/or cultivation medium of the plant in a non-invasive manner (e.g., spraying, air-aspiration inoculation, soaking, etc.) or an invasive manner (e.g., via incision or wound, etc.), thereby improving the ability of the plant to resist biotic or abiotic stress.
The present invention described herein for enhancing the stress tolerance of a plant may include, but is not limited to, enhancing the plant's ability to resist biotic or abiotic stress.
In the above examples, the kind of the plant is not particularly limited. However, in some examples, the aforementioned plant may be, for example, a dicot or a monocot, wherein the dicot may be, for example, a solanaceous plant or a cucurbitaceous plant, a specific example of the solanaceous plant may be tomato, a specific example of the cucurbitaceous plant may be papaya, and a specific example of the monocot may be rice.
In the above embodiments, the kind of the cultivation medium is not particularly limited. However, in some examples, specific examples of the aforementioned cultivation medium may include, but are not limited to, water, soil, culture soil, foamed stone, bark, artificial soil, technical soil, vermiculite, perlite, sawdust, zeolite, aquatic weed, or any combination thereof.
The biological stress described herein may include infection with pathogenic bacteria, but the type of pathogenic bacteria varies depending on the plant species, and the present invention is not particularly limited. In one example, the pathogenic bacteria may include, but are not limited to, anthrax and Pseudomonas, wherein specific examples of anthrax may include, but are not limited to, Colletotrichumgloeosporioides (colletotrichum gloeosporioides), and specific examples of Pseudomonas may include, but are not limited to, Pseudomonas syringae (Pseudomonas syringae pv. tomato) and Pseudomonas syringae, a nonpathogenic variety.
The abiotic stress conditions described herein may include various environments not suitable for plant growth, and specific examples thereof may include, but are not limited to, high temperatures of at least 45 ℃, ultraviolet light, and drought for at least 14 days, although the invention is not limited thereto.
The improvement of the stress tolerance of the plant according to the present invention may include, but is not limited to, the improvement of the stress tolerance of the plant in the future by improving the ability of rhizobacteria to entangle the roots of the plant, increasing the hydrogen peroxide content and the peroxidase activity in the plant, and improving the expression of the disease resistance gene of the plant (for example, promoting the expression of Jasmonic Acid (JA) and Salicylic Acid (SA) related genes in the plant).
In an embodiment of the present invention, the dosage form of the plant growth regulator can be, for example, liquid, powder, paste, block, lozenge, capsule, or combined with other suitable carriers, but the present invention is not limited thereto. In addition, the plant growth regulator uses lactobacillus fermentation culture solution as an effective component, and other various conventional inactive components can be selectively added according to actual requirements.
In one embodiment of the present invention, the concentration of the plant growth regulator is not particularly limited, and may be adjusted depending on the type of plant and the site of application. For example, in the case of applying to the leaf blade, the fermentation broth of Lactobacillus (strain concentration 10) can be administered by spraying the surface of the leaf blade (10-fold dilution of the stock solution) at least 10 times, or by inoculating the surface of the leaf blade by suction7CFU/mL). In the case of application to soil, a single effective dose thereof may be, for example150mL of a fermentation culture of Lactobacillus was administered to 2kg of soil (stock solution diluted 10-fold), but the present invention is not limited to the examples set forth herein.
The present invention is described in detail by the following embodiments, which are not intended to limit the scope of the invention, and it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
EXAMPLE one preparation of plant growth regulators from Lactobacillus paracasei GMNL-32
First, this example evaluates the bacteriological characteristics of Lactobacillus paracasei (Lactobacillus paracasei; GMNL-32).
1. Bacteriological characteristics of Lactobacillus paracasei GMNL-32
The source of Lactobacillus paracasei (Lactobacillus paracasei) GMNL-32 is the healthy human gastrointestinal tract, and the appearance and physiological characteristics thereof are disclosed in Chinese patent publication No. CN 100396771C, which is incorporated herein by reference.
Secondly, after the Lactobacillus paracasei GMNL-32 is analyzed by API 50 CHI V5.1 identification kit (not shown in the figure), the Lactobacillus paracasei is identified.
In addition, after total RNA of Lactobacillus paracasei GMNL-32 was extracted by a conventional method, a partial sequence of the 16S rDNA gene was amplified by a primer pair (upstream primer PAF primer and downstream primer 536R primer) having the sequence numbers (SEQ ID NO):1 and 2, and the resulting nucleic acid fragment was represented by the sequence number (SEQ ID NO): 3. Methods for extracting total RNA are well known to those skilled in the art to which the present invention pertains, and are not described herein.
The nucleic acid fragment was confirmed to be Lactobacillus paracasei when it was aligned with the National Center for Biotechnology Information (NCBI) gene bank (GenBank).
2. Preparation of plant growth regulator
After culturing Lactobacillus paracasei GMNL-32 in a 5L fermenter overnight, the fermentation broth (about 10 bacteria count) was collected6CFU/mL to about 109CFU/mL) is the stock solution of the lactobacillus paracasei GMNL-32 fermentation culture.
And (3) carrying out heat killing on the stock solution for 15 to 30 minutes at a high temperature (90 to 121 ℃) to obtain the heat-killed fermentation culture solution for the lactobacillus paracasei GMNL-32.
After the fermentation culture stock solution is diluted by 10 times by water, the fermentation culture solution is respectively a lactobacillus paracasei GMNL-32 fermentation culture solution (hereinafter referred to as GMNL-32) or a lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution (hereinafter referred to as GMNL-32_ HK) so as to further evaluate the effects of the two as plant growth regulators.
Example II evaluation of the Effect of plant growth regulators on improving the stress resistance of plants
1. Evaluation of the Effect of plant growth regulators on improving the resistance of tomato plants to biotic stress
1.1 inoculation of plant pathogenic bacteria
In this example, tomato plants (Solanum lycopersicum, line: Nongyou 301, Nongyou Miao, Taiwan), 4 to 5 weeks old) were used, and leaves thereof were treated in the field with the fermentation culture solution GMNL-32 of Lactobacillus paracasei or the fermentation culture solution GMNL-32 of Lactobacillus paracasei of example I for 11 times, and then inoculated with the following plant pathogenic fungi, respectively.
Colletotrichum gloeosporioides (tomato anthracnose) WAs cultured in 1/5 Potato-glucose-Agar medium (PDA) and the number of conidia WAs counted by a hemocytometer after shaking 5 minutes in 5mL of 0.1% water Agar (Wateragar; WA) medium containing 0.05% Tween 20. Next, the conidia were adjusted to a concentration of 106spores/mL [ containing 1/5 potato-glucose-Broth (PDB) and 10mM MgSO4) Spraying and inoculating on tomato leaves treated by the fermentation culture solution of lactobacillus paracasei GMNL-32 or the heat-killed fermentation culture solution of lactobacillus paracasei GMNL-32. After 5 days of inoculation, the appearance of the plants was photographed and the number of lesions per leaf was counted, wherein the dark gangrene of the leaf represents one lesion, and the results are shown in FIG. 1A and FIG. 1B.
In addition, Pseudomonas syringae pv. tomatoo DC 3000; PstDC 3000; causing tomato bacterial spot disease) And Pseudomonas syringae Pst DC3000avrRpt2 without pathogenic variants were cultured in King's medium B containing 50ppm of rifamycin (rifampicin); KBM) and KBM containing 50ppm of lipomycin and kanamycin, the concentration of the bacterial liquid was adjusted to 10 with Reverse Osmosis (RO) water7CFU/mL, inoculated on tomato leaves treated by the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution by an air-bleed inoculation method. 4 days after inoculation, the appearance of the plants was photographed and the number of lesions per leaf was calculated, the results of which are shown in FIGS. 2A to 2D.
The data obtained above are all expressed as mean ± Standard Deviation (SD), and statistical analysis is performed by using commercially available statistical software (for example, SPSS 12.0), group differences are compared by one-way analysis of variance, and significant difference comparison is performed by duncan's multiple range method. The english letters a, B, and c in fig. 1B, 2B, and 2D represent statistical results, respectively, and different letters indicate that there is a significant difference between groups (p <0.05), and the same letter indicates that there is no statistical difference between groups (p > 0.05).
1.2 evaluation of the Effect of plant growth regulators on improving the Anthrax bacteria resistance of tomato plants
Referring to fig. 1A and 1B, there are shown leaf appearance (fig. 1A) and number histogram of lesion number per leaf of colletotrichum gloeosporioides inoculated to tomato plants according to an embodiment of the present invention after administration of plant growth regulator. In FIG. 1B, Mock treatment (Mock) represents the appearance of leaves of plants not given plant growth regulator but inoculated with colletotrichum gloeosporioides.
The results of the sham-treated groups of FIGS. 1A and 1B show that the leaves of tomato sprayed with colletotrichum gloeosporioides for 5 days did yellow (as shown in FIG. 1A) and black rotten gangrene spots (FIGS. 1A and 1B) were produced.
However, the results of the GMNL-32 or GMCL-32_ HK treated groups of FIGS. 1A and 1B show that, after the tomato leaves were sprayed with colletotrichum gloeosporioides for 5 days after the plant growth regulator was administered to the tomato leaves, the leaves did not yellow (as shown in FIG. 1A), and the number of lesions was significantly reduced (FIGS. 1A and 1B), showing that the GMNL-32 fermentation culture of Lactobacillus paracasei could increase the effect of the tomatoes against colletotrichum gloeosporioides infection.
Secondly, the lactobacillus paracasei GMNL-32 fermentation culture solution still has the protection effect after heat killing treatment (namely GMCL-32_ HK) (as shown in figure 1B), and has no statistically significant difference (the same letters indicate no statistical difference (p >0.05) among groups) compared with the lactobacillus paracasei GMNL-32 fermentation culture solution, and the protection effect of the lactobacillus paracasei GMNL-32 fermentation culture solution is not influenced by heat.
1.3 evaluation of the Effect of plant growth regulators on improving Pseudomonas resistance in tomato plants
Please refer to fig. 2A to 2D, which show the appearance of leaves (fig. 2A, 2C) and the number of lesions per leaf histogram (fig. 2B, 2D) of a tomato plant inoculated with pseudomonas syringae after administration of a plant growth regulator according to an embodiment of the present invention. In FIGS. 2A-2D, Mock treatment (Mock) represents the appearance of the leaves of a plant not administered with a plant growth regulator but inoculated with either Pseudomonas syringae tomato (FIGS. 2A-2B) or Pseudomonas syringae tomato of the avirulent variety (FIGS. 2C-2D).
The results of the sham-treated groups of FIGS. 2A-2D show that the inoculation of tomato leaves with Pseudomonas syringae (PstDC3000) or non-pathogenic variety of Pseudomonas syringae (Pst DC3000avrRpt 2) for 4 days did result in yellowing of the leaves (as shown in FIG. 2A) and the development of black rot gangrene spots (FIGS. 2A-2D).
The results of the GMNL-32 treated groups in FIGS. 2A to 2D show that the number of leaf lesions is significantly reduced after 4 days of inoculation of tomato leaves with either Pseudomonas syringae (Pst DC3000) or the non-pathogenic variety of Pseudomonas syringae (pstDC3000 avrRpt2) after administration of plant growth regulator (FIGS. 2B and 2D), showing that the effect of the tomatoes against infection with tomato Pseudomonas syringae can be increased by the Lactobacillus paracasei GMNL-32 fermentation broth.
Secondly, the lactobacillus paracasei GMNL-32 fermentation broth after heat killing treatment (i.e. GMCL-32_ HK) still has a protective effect (as shown in fig. 2B and fig. 2D), and has no statistically significant difference (same letters indicate no statistical difference (p >0.05) between groups) compared with the lactobacillus paracasei GMNL-32 fermentation broth, which means that the protective effect of the lactobacillus paracasei GMNL-32 fermentation broth is not affected by heat.
2. Evaluation of the Effect of plant growth regulators on improving the resistance of tomatoes to abiotic stress
In this example, tomato plants (Solanum lycopersicum, strain: Nongyou 301, Nongyou Miao, Taiwan), 4 to 5 weeks old were treated with RO water (Mock), the fermentation culture solution of Lactobacillus paracasei GMNL-32 according to example, or the fermentation culture solution of Lactobacillus paracasei GMNL-32 by thermal sterilization (irrigation) for 9 days, and then subjected to the following various adversity treatments.
2.1 evaluation of the Effect of plant growth regulators on improving the Heat resistance of tomato plants
The tomato plants were treated at 45 ℃ for 6 hours to achieve high temperature injury, the appearance of the plants was photographed, and the injury degree was observed in a graded manner in the next 24 hours, with the results shown in fig. 3A and 3B. The damage degree is graded and quantified by the formula (I):
high temperature damage (%) [ (number of curled leaves/total number of leaves) × 100% ]. (I)
The english letters a, B, c in fig. 3B represent the statistical results, respectively, and different letters indicate significant differences between groups (p < 0.05).
Referring to fig. 3A and 3B, there are shown the appearance of plants heat-treated with a plant growth regulator (fig. 3A) and a histogram of heat damage (fig. 3B) in tomato plants according to an embodiment of the present invention. In FIG. 3B, the vertical axis represents the thermal damage rate (%), and the horizontal axis represents each treatment group, wherein Mock treatment (Mock) represents treatment with water, GMNL-32 represents treatment with Lactobacillus paracasei GMNL-32 fermentation broth, and GMNL-32_ HK represents treatment with Lactobacillus paracasei GMNL-32 heat-killed fermentation broth.
As shown in FIG. 3A, the tomato plants in the sham-treated group were treated with water, then at 45 ℃ for 6 hours and left for 24 hours, and the leaves were curled and died.
However, the results of the GMNL-32 or GMCL-32_ HK treated groups of FIGS. 3A and 3B showed that leaf curl was less severe in tomato plants after pouring with the Lactobacillus paracasei GMNL-32 fermentation broth or the Lactobacillus paracasei GMNL-32 heat-killed fermentation broth (as shown in FIG. 3A). Furthermore, the results of fig. 3B, which quantitatively show that the thermal injury rate was graded, show that the thermal injury rate of tomato plants was significantly reduced by the treatment with the lactobacillus paracasei GMNL-32 fermentation broth or the heat-killed fermentation broth of lactobacillus paracasei GMNL-32, and that the heat-resistance of tomato plants was increased by the lactobacillus paracasei GMNL-32 fermentation broth.
Secondly, the protection effect (shown in figure 3B) of the lactobacillus paracasei GMNL-32 fermentation culture solution is still achieved after heat killing treatment (namely GMCL-32_ HK), which represents that the protection effect of the lactobacillus paracasei GMNL-32 fermentation culture solution is not affected by heat.
2.2 evaluation of the Effect of plant growth regulators on reducing UV damage to tomato plants
After irradiating the tomato plants with UV-C (253.7 nm wavelength) for 30 minutes to ultraviolet damage, the appearance of the plants was photographed for the next 24 hours and the damage degree was graded by the formula (II), and the results are shown in FIG. 4A and FIG. 4B:
percent damage [% ]
(N0*n+N1*n+N2*n+N3*n)/(Nt*nt)*100。 (II)
In formula (II), N represents the number of stages, N represents the number of samples, Nt represents the total number of stages, and Nt represents the total number of samples. The English letters a, B, c in FIG. 4B represent the statistical results, respectively, different letters indicate significant difference (p <0.05) among groups, and the same letters indicate no statistical difference (p >0.05) among groups.
Referring to fig. 4A and 4B, there are shown the appearance of plants irradiated with ultraviolet rays (fig. 4A) and a histogram of thermal damage (fig. 4B) after a tomato plant is administered with a plant growth regulator according to an embodiment of the present invention. In FIG. 4B, the vertical axis represents the UV damage ratio (%), and the horizontal axis represents each treatment group, wherein Mock treatment (Mock) represents treatment with water, GMNL-32 represents treatment with Lactobacillus paracasei GMNL-32 fermentation broth, and GMNL-32_ HK represents treatment with Lactobacillus paracasei GMNL-32 heat-killed fermentation broth.
As shown in FIG. 4A, it was confirmed that the tomato plants in the sham-treated group were irradiated with UV-C (253.7 nm wavelength) for 30 minutes after water treatment and left for 24 hours, and the leaves were curled and died.
However, the results of the GMNL-32 or GMCL-32_ HK treated groups of FIGS. 4A and 4B showed that leaf curl was significantly reduced in tomato plants after pouring with the Lactobacillus paracasei GMNL-32 fermentation broth or the Lactobacillus paracasei GMNL-32 heat-killed fermentation broth (as shown in FIG. 4A). Moreover, the results of fig. 4B, which quantitatively show that the heat damage rates were graded, show that the ultraviolet damage levels of tomato plants were significantly reduced after treatment with the lactobacillus paracasei GMNL-32 fermentation broth or the lactobacillus paracasei GMNL-32 heat-killed fermentation broth, compared to the ultraviolet damage levels of the sham-treated groups, and that the ultraviolet resistance of tomato plants was increased by the lactobacillus paracasei GMNL-32 fermentation broth.
Secondly, the lactobacillus paracasei GMNL-32 fermentation broth after heat killing treatment (i.e. GMCL-32 — HK) still has a protective effect (as shown in fig. 4B), and has no statistically significant difference (the same letters indicate no statistical difference (p >0.05) between groups) compared with the lactobacillus paracasei GMNL-32 fermentation broth), which means that the protective effect of the lactobacillus paracasei GMNL-32 fermentation broth is not affected by heat.
2.3 evaluation of the Effect of plant growth regulators on reducing drought-damaged tomato plants
The drought damage method comprises treating tomato plant with Lactobacillus paracasei GMNL-32 fermentation culture solution or Lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution, stopping watering, photographing plant appearance in 14 days, and grading damage degree with formula (II), with the results shown in FIG. 5A and FIG. 5B.
The english letters a, B, c in fig. 5B represent statistical results, respectively, with different letters indicating significant difference (p <0.05) among groups and the same letters indicating no statistical difference (p >0.05) among groups.
Please refer to FIGS. 5A and 5B, which show the appearance of drought-treated tomato plants (FIG. 5A) and the histogram of drought damage (FIG. 5B) after administration of plant growth regulator. In FIG. 5B, the vertical axis represents the drought damage rate (%), and the horizontal axis represents each treatment group, where Mock treatment (Mock) represents treatment with water, GMNL-32 represents treatment with Lactobacillus paracasei GMNL-32 fermentation broth, and GMNL-32_ HK represents treatment with Lactobacillus paracasei GMNL-32 heat-killed fermentation broth.
As shown in FIG. 5A, the tomato plants in the sham-treated group were treated with water and then cut off for 14 days, and the leaves were curled and died.
However, the results of the GMNL-32 or GMCL-32_ HK treated groups of FIGS. 5A and 5B showed that leaf blight was significantly reduced when tomato plants were watered with the Lactobacillus paracasei GMNL-32 fermentation broth or the Lactobacillus paracasei GMNL-32 heat-killed fermentation broth (as shown in FIG. 5A). And the results of grading and quantifying the drought damage rate in fig. 5B show that the tolerance of tomato plants to drought can be significantly increased after treatment with the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution compared to the drought damage degree of the sham-treated group.
Secondly, the lactobacillus paracasei GMNL-32 fermentation broth after heat killing treatment (i.e. GMCL-32 — HK) still has a protective effect (as shown in fig. 5B), and has no statistically significant difference (the same letters indicate no statistical difference (p >0.05) between groups) compared with the lactobacillus paracasei GMNL-32 fermentation broth), which means that the protective effect of the lactobacillus paracasei GMNL-32 fermentation broth is not affected by heat.
3. Evaluation of Effect of plant growth regulator on improving ability of rhizobacteria to gather tomato roots
It is known that Plant Growth Promoting Rhizobacteria (PGPR) include Bacillus thuringiensis (Bacillus thuringiensis) and Bacillus amyloliquefaciens (B. amyloliquefaciens). This example evaluates the ability of plant growth regulators to regulate the root entanglement of rhizobacteria, thereby improving the plant's resistance to stress.
Firstly, bacillus thuringiensis and liquefied starch sporeThe Bacillus was inoculated into Nutrient Broth (NB) and cultured at 28 ℃ for 14 hours and 16 hours. Then, the concentration of the resulting bacterial liquid was adjusted to OD600nmAfter 0.8, the mixture was poured into 2kg of soil (soil bacteria count: 10)8CFU/g) and 150mL of Lactobacillus paracasei GMNL-32 fermentation culture solution or Lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution are added, tomato root tissues are collected for 1, 5, 9, 13, 17 and 30 days, after weighing, 20mL of 0.1% WA containing 0.025% SILWET L-77 is shaken for 5 minutes, and then the tomato root tissues are sequentially diluted and coated on an LB (Luria-Bertaini) culture medium by glass beads, and after culturing for 1 day at 28 ℃, the colony number is calculated to evaluate the entanglement capacity of rhizobacteria on plant roots, and the results are respectively shown in FIG. 6A and FIG. 6B. The English letters a, B, and c in FIG. 6A and FIG. 6B represent the statistical results, respectively, and different letters indicate significant difference (p) between groups<0.05), same letter indicates no statistical difference (p) between groups>0.05)。
Please refer to fig. 6A and 6B, which show histograms of the number of root bacteria of tomato plants inoculated with the bacterial solution of bacillus thuringiensis (fig. 6A) or bacillus amyloliquefaciens (fig. 6B) and then administered with plant growth regulators according to an embodiment of the present invention. In FIGS. 6A and 6B, Mock treated (Mock) group represents the number of root bacteria to which the Bacillus thuringiensis or Bacillus amyloliquefaciens inoculated bacterial solution was administered with an equal volume of water only (but no plant growth regulator).
The results of the sham-treated group of fig. 6A show that after 9 days, about 100-fold decrease in the number of bacteria was observed after the tomato roots were inoculated with the bacillus thuringiensis solution. However, if the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution is added at the same time when the bacterial solution is inoculated, the reduction of the bacterial count of the bacillus thuringiensis can be slowed down, and the bacterial count of the bacillus thuringiensis can be maintained for 30 days.
As shown in the results of the dummy treatment group of FIG. 6B, it was found that the number of bacteria was decreased by about 100 times after 5 days after inoculating the roots of the tomatoes with the liquid of Bacillus amyloliquefaciens. However, if the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution is added during the inoculation of the bacterial solution, the reduction of the number of the liquefied bacillus amyloliquefaciens can be slowed down, and the number of the liquefied bacillus amyloliquefaciens can be maintained for 21 days, which shows that the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution has the effect of maintaining plant rhizosphere bacteria to be entangled at the roots of tomato plants.
Secondly, the lactobacillus paracasei GMNL-32 fermentation culture solution still has the protection effect (as shown in figures 6A and 6B) after being thermally killed (namely GMCL-32_ HK), which means that the protection effect of the lactobacillus paracasei GMNL-32 fermentation culture solution is not influenced by heat.
4. Evaluation of Effect of plant growth regulator on increasing Hydrogen peroxide content in tomato
After the tomato plants are transplanted and grown for 5 days, 25mL of lactobacillus paracasei GMNL-32 fermentation culture solution or lactobacillus paracasei GMNL-32 heat-killing fermentation culture solution is poured at the roots. After 0, 12, 24, 36 and 48 hours of treatment, 0.05g of the second leaf at the top of the plant was taken, ground with liquid nitrogen, and shaken for 10 seconds with 600. mu.L of phosphate buffer (50mM, pH 6.8). Then, the mixture was centrifuged at 9,000Xg for 25 minutes, and 400. mu.L of the supernatant solution was added to 200. mu.L of a titanium sulfate solution, followed by shaking for 10 seconds and centrifugation at 6,000Xg for 15 minutes. Then, 200. mu.L of the supernatant solution was taken, absorbance at 410nm was measured, and the hydrogen peroxide content was calculated by using the formula (III), and the result is shown in FIG. 7:
hydrogen peroxide content ═ hydrogen peroxide
{[(OD410nm-0.0011)/0.0004]1.5 }/weight (mg). (III)
The English letters a and b in FIG. 7 represent the statistical results, respectively, different letters indicate that there is a significant difference between groups (p <0.05), and the same letters indicate that there is no statistical difference between groups (p > 0.05).
Referring to fig. 7, there is shown a histogram of leaf hydrogen peroxide content of tomato plants given plant growth regulator in accordance with one embodiment of the present invention. In FIG. 7, the Mock treated (Mock) group represents the leaf hydrogen peroxide content given only an equal volume of water (but no plant growth regulator).
The results of the sham-treated group of fig. 7 show that the hydrogen peroxide content of the leaves did not change significantly after the tomato plants were treated with water. However, the hydrogen peroxide content of the leaves can be effectively improved after the tomato plants are treated by the lactobacillus paracasei GMNL-32 fermentation culture solution for 12 hours. In addition, the hydrogen peroxide content of the leaves is effectively improved after the treatment of the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution for 12 hours and 24 hours, and the effect of improving the hydrogen peroxide content of the tomatoes is shown in the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution.
5. Evaluation of the Effect of plant growth regulators on increasing peroxidase Activity of tomato
After the tomato plants are transplanted and grown for 5 days, 25mL of lactobacillus paracasei GMNL-32 fermentation culture solution or lactobacillus paracasei GMNL-32 heat-killing fermentation culture solution is poured at the roots. After 12, 24, 36 and 48 hours of treatment, 0.05g of the second leaf at the top of the plant was taken, ground with liquid nitrogen, and shaken for 10 seconds with 1,000. mu.L of phosphate buffer solution (50mM, pH 5.8). Then, the mixture was centrifuged at 12,000Xg for 20 minutes, 10. mu.L of the supernatant was taken, 100. mu.L of phosphate buffer solution, 100. mu.L of phosphomethoxyphenol and 90mL of 39mM hydrogen peroxide were added, the absorbance at 470nm was measured, and the peroxidase (peroxosidase) activity was calculated using the formula (IV), and the results are shown in FIG. 8:
peroxidase activity (. DELTA.abs 470nm/min/gFW) ═
(△OD470nm) Reaction volume (mL)/dilution rate/reaction time/weight (g). (IV)
The English letters a and b in FIG. 8 represent the statistical results, respectively, different letters indicate that there is a significant difference between groups (p <0.05), and the same letters indicate that there is no statistical difference between groups (p > 0.05).
Referring to FIG. 8, there is shown a histogram of the leaf catalase activity content of tomato plants given plant growth regulator according to an embodiment of the present invention. In FIG. 8, the Mock treated (Mock) group represents leaf peroxidase activity given only an equal volume of water (but no plant growth regulator).
The results of the sham-treated group of fig. 8 show that the peroxidase activity of the leaf portions did not change significantly after the tomato plants were treated with water. However, compared to the Mock-treated (Mock) group, the peroxidase activity of the leaf of tomato plants was effectively increased 5-fold after 24 hours of treatment with the fermentation broth of Lactobacillus paracasei GMNL-32 and maintained for up to 48 hours. In addition, after the treatment of the heat-killed fermentation culture solution of lactobacillus paracasei GMNL-32 for 12 hours, the peroxidase activity of the leaves can be effectively improved and maintained for 36 hours, and the effect of improving the hydrogen peroxide content of the tomatoes is shown in the heat-killed fermentation culture solution of lactobacillus paracasei GMNL-32 or the heat-killed fermentation culture solution of lactobacillus paracasei GMNL-32.
6. Evaluation of Effect of plant growth regulator on improving expression of disease-resistant Gene of tomato
It is known that regulatory genes (e.g., LeOPR3, LeCOI1 and LeJAZ1) in a signal transmission path related to Jasmonic Acid (JA) and regulatory genes (e.g., LePR1) in a signal transmission path related to salicylic acid (salicylic acid; SA) are related to plant disease resistance, and the effect of a plant growth regulator on tomato disease resistance is evaluated by detecting the mRNA expression level of the genes.
After tomato plants are cultured for 20 days, 25mL of lactobacillus paracasei GMNL-32 fermentation culture solution or lactobacillus paracasei GMNL-32 heat-killing fermentation culture solution is poured at roots. After 1 and 3 days of treatment, the second leaf from the top of the plant is taken and total RNA from plant tissue is extracted using a commercially available Total RNA extraction kit (e.g., tissue Total RNA mini kit). Next, 1. mu.L of RNA was Reverse transcribed into cDNA using a commercially available Reverse Transcription kit (e.g., MMLV Reverse Transcription kit), 1. mu.L of 10mM primer pair, 2. mu.L of 10 XPCR buffer, 1. mu.L of 2.5mM dNTP, 0.1. mu.L of 5 units/. mu.L Pro TaqDNA polymerase (polymerase), and 10. mu.L of 2 XPER SYBR Green PCR Master Mix were added, and the total volume of the reaction was made up to 20. mu.L with sterile water, and a real-time Quantitative (Quantitative real time) PCR reaction was performed, the results of which are shown in FIGS. 9A to 10B.
The primer pair comprises an upstream primer and a downstream primer of LeOPR3, LeCOI1, LeJAZ1 and LePR1, the sequence of the upstream primer and the downstream primer is shown in sequence numbers (SEQ ID NO): 4-13, the ratio of the PCR reaction cycle numbers of LeOPR3, LeCOI1, LeJAZ1 and LePR1 to the PCR reaction cycle number of ef1 α is used as relative value data.
6.1 evaluation of the Effect of plant growth regulators on increasing jasmonic acid-related signaling pathway in tomato
Please refer to fig. 9A to 9F, which show the mRNA relative content histograms of the genes LeOPR3, LeCOI1 and LeJAZ1 after the tomato plants according to an embodiment of the present invention are administered with the plant growth regulator.
As shown in fig. 9A to 9F, the expression levels of mRNA of LeOPR3, LeCOI1, and LeJAZ1 in the Mock-treated group were 1.0, and the expression levels of JA regulatory genes LeOPR3, LeCOI1, and LeJAZ1 in the leaves of tomato plants treated for 1 day with the lactobacillus paracasei GMNL-32 fermentation broth (shown in fig. 9A to 9C) or the lactobacillus paracasei GMNL-32 heat-killed fermentation broth (shown in fig. 9D to 9F) were significantly higher than those in the Mock-treated group (Mock). On the other hand, the expression level of LeCOI1 in tomato plants treated with the lactobacillus paracasei GMNL-32 fermentation culture solution (shown in fig. 9A to 9C) or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution (shown in fig. 9D to 9F) after 3 days of treatment was significantly different from that in the Mock-treated group (Mock), indicating that the treatment of the plants with the lactobacillus paracasei GMNL-32 fermentation culture solution could indeed activate the disease-resistant pathway related to Jasmonic Acid (JA) in plants.
6.2 evaluation of the Effect of plant growth regulators on increasing the salicylic acid-related signalling pathway in tomatoes
Referring to FIGS. 10A and 10B, there are shown histograms of the relative content of mRNA of the gene LePR1 after tomato plants are administered with a plant growth regulator according to an embodiment of the present invention.
As shown in fig. 10A and 10B, the mRNA expression level of LePR1 in the Mock-treated group was 1.0, and the expression level of the SA regulatory gene LePR1 in the leaf of each tomato plant was significantly higher than that in the Mock-treated group (Mock) after 1 day of treatment with the lactobacillus paracasei GMNL-32 fermentation broth (shown in fig. 10A) or the lactobacillus paracasei GMNL-32 heat-killed fermentation broth (shown in fig. 10B). After 3 days of treatment, the expression level of LePR1 in tomato plants treated by the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution (shown in figure 10B) is still significantly different from that of a sham-treated group (Mock), which shows that the disease-resistant path related to Salicylic Acid (SA) in the plants can be indeed activated by treating the plants with the lactobacillus paracasei GMNL-32 fermentation culture solution.
The results show that the tomato is used as a plant model, and the tomato plant model proves that the tomato plant biological stress resistance (comprising the tomato plant infection of colletotrichum gloeosporioides Pst DC3000avrRpt2 inhibiting colletotrichum gloeosporioides, tomato pseudomonas syringae Pst DC3000 and non-pathogenic variety tomato pseudomonas syringae Pst DC3000avrRpt 2) and the abiotic stress resistance (comprising high temperature, ultraviolet ray and drought) of the plant are improved after the treatment of the fermentation culture solution of lactobacillus paracasei GMNL-32 or the heat killing fermentation culture solution of lactobacillus paracasei GMNL-32. The improvement of the disease resistance of the plant is possible to increase the resistance to the biological property (i.e. pathogenic bacteria) and the abiotic property (i.e. environment) by maintaining the capability of rhizobacteria to be entangled with the roots of the plant, effectively increasing the hydrogen peroxide content and the peroxidase activity in the plant body and massively expressing genes related to the disease resistance (comprising regulatory genes LeOPR3, LeCOI1 and LeJAZ1 in a jasmonic acid-related signal transmission path and a regulatory gene LePR1 in a salicylic acid-related signal transmission path), so that the plant is more capable of adapting to the adverse environment or climate change.
It is added that the lactobacillus paracasei GMNL-32 used in the invention is derived from human gastrointestinal tract, and the lactobacillus paracasei GMNL-32 fermentation culture solution or the lactobacillus paracasei GMNL-32 heat-killed fermentation culture solution has excellent heat stability (the protection effect is not influenced by heat), is safe and has no side effect through the experiment, and has potential application to plant growth regulators or plant liquid fertilizers containing the plant growth regulators.
In summary, although the present invention is described with reference to specific strains, specific formulations, specific subjects, specific administration forms, or specific evaluation forms as examples, the plant growth regulator for improving stress tolerance of the present invention and the use thereof are described, but those skilled in the art will recognize that the present invention is not limited thereto, and that the present invention may be carried out using other strains, other formulations, other subjects, other administration forms, or other evaluation forms without departing from the spirit and scope of the present invention.
For example, the plant growth regulator of the present invention may be administered to the whole plant, a part or a culture medium thereof of other dicotyledonous plants (e.g., papaya of the family citrullinaceae) or monocotyledonous plants (e.g., rice) to increase the plant's ability to resist biotic and abiotic stress. In addition, the dosage form of the plant growth regulator of the present invention may be liquid, powder, paste, block, tablet, capsule, or combined with other suitable carriers, but the present invention is not limited thereto.
The embodiments show that the plant growth regulator for improving stress tolerance and the application thereof have the advantages that the plant growth regulator consists of the fermentation culture solution of lactobacillus paracasei, has excellent thermal stability, safety and no side effect, can obviously improve the biological stress tolerance and the abiotic stress tolerance of plants, and can be further used as the plant growth regulator or the application for preparing the composition for improving the plant stress tolerance.
While the invention has been described with respect to various embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
[ biological Material Collection ]
Lactobacillus paracasei GMNL-32 (also called GM-080) is preserved in the type culture preservation center (CCTCC) of Wuchang Lojia mountain Wuhan university in Wuhan city, Hubei province, China, with the preservation date of 2004 for 2 months and 19 days and the preservation number of CCTCC M204012.
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Claims (13)
1. The use of a Lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance, comprising administering a Lactobacillus fermentation broth to the whole plant, a plant part and/or a cultivation medium of the plant to enhance the plant's ability to resist biotic or abiotic stress, wherein the Lactobacillus fermentation broth is derived from Lactobacillus paracasei (Lactobacillus paracasei) GMNL-32, and the Lactobacillus paracasei GMNL-32 is preserved in ccm collection (CCTCC) of wuchang mountain laohanshan university, martin north of lake, 2 months 19, under the preservation number cct 204012, and the Lactobacillus fermentation broth is a live or dead bacterium comprising the Lactobacillus paracasei GMNL-32.
2. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance according to claim 1, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.
3. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance as claimed in claim 2, wherein the dicotyledonous plant is a solanaceae plant or a citrullinaceae plant.
4. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance as claimed in claim 3, wherein the solanaceous plant is tomato.
5. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance as claimed in claim 3, wherein the melon plant is papaya.
6. Use of a lactobacillus fermentation broth according to claim 3 for the preparation of a plant growth regulating composition for enhancing stress tolerance, wherein the monocot is rice.
7. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance according to claim 1, wherein the plant parts comprise a leaf, a stem and/or a root.
8. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance as claimed in claim 1, wherein the cultivation medium is selected from the group consisting of water, soil, culture soil, foaming condensed stone, bark, artificial soil, technical soil, vermiculite, perlite, snake wood chips, zeolite, waterweed and any combination thereof.
9. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition with enhanced stress tolerance according to claim 1, wherein the biological stress comprises infection by a pathogenic bacterium.
10. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition with enhanced stress tolerance according to claim 9 wherein the pathogenic bacteria comprise anthrax and pseudomonas.
11. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition with improved stress tolerance according to claim 10, wherein the anthrax genus comprises Colletotrichumgloeosporioides (Colletotrichumgloeosporioides).
12. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance according to claim 10, wherein the pseudomonas comprises pseudomonas lycopersici (pseudomonas assaynyingae pv. tomato) and pseudomonas lycopersici without pathogenic varieties.
13. Use of a lactobacillus fermentation broth for the preparation of a plant growth regulating composition for enhancing stress tolerance according to claim 1, wherein the abiotic stress comprises high temperature of at least 45 ℃, uv light and drought for at least 14 days.
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